Left atrial epicardial adipose tissue is closely associated with left atrial appendage flow velocity in patients with nonvalvular atrial fibrillation

Epicardial adipose tissue (EAT) can play an important role in the occurrence and development of atrial fibrillation and stroke. In this study, we explored the relationship between left atrial epicardial adipose tissue (LA-EAT) and left atrial appendage flow velocity (LAA-FV) in patients with nonvalvular atrial fibrillation (NV-AF). A total of 145 patients with NV-AF who underwent their first radiofrequency ablation were enrolled. They underwent left aortopulmonary vein computed tomography angiography (CTA) and transesophageal echocardiography (TEE) before AF ablation. Left atrial (LA) electroanatomical mapping was performed intraoperatively to assess left atrial voltage. Univariate regression analysis showed that LAA-FV was lower in patients with a low voltage zone (LAA-FV; 35.02 ± 10.78 cm/s vs. 50.60 ± 12.17 cm/s, P < 0.001). A multiple linear regression model showed that the left atrial low voltage zone (β = − 0.311 P < 0.001), LA-EAT volume (β = − 0.256 P < 0.001), left atrial appendage shape (β = − 0.216 P = 0.041), LAVI (β = − 0.153 P = 0.041), and type of atrial fibrillation (paroxysmal vs. persistence) (β = − 0.146 P < 0.048) were independent predictors of LAA-FV. In NV-AF patients, the increase in LA-EAT volume is related to the decrease in LAA-FV.

www.nature.com/scientificreports/ Methods Study population. A total of 145 patients with nonvalvular atrial fibrillation who underwent their first radiofrequency ablation at the Affiliated Hospital of Xuzhou Medical University between 2020 and 2021 were selected. All patients underwent CTA and TEE examinations of the left atrium and pulmonary vein before the operation. There were 76 patients with paroxysmal atrial fibrillation and 69 patients with nonparoxysmal atrial fibrillation. All patients were diagnosed with atrial fibrillation by electrocardiogram and/or dynamic electrocardiogram. Paroxysmal atrial fibrillation is defined as atrial fibrillation that lasts less than 7 days and terminates spontaneously, while persistent atrial fibrillation is defined as atrial fibrillation that lasts more than 7 days or requires electrical cardioversion and/or drug cardioversion to terminate. Exclusion criteria: (1) previous catheter ablation of atrial fibrillation or other cardiac surgery; (2) hyperthyroidism; (3) rheumatism and other cardiac valvular diseases; (4) active connective tissue disease; and (5) inability to undergo CTA or CT imaging or inability to obtain complete left atrial appendage data because of contrast medium allergy. This study was reviewed by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University, and all patients signed informed consent forms.

Echocardiographic measurements. Transthoracic echocardiography (TTE) was performed with a
Philips EPIQ7c ultrasonic diagnostic instrument, S5-1 probe and probe frequency 1-5 MHz. Parameters such as left ventricular ejection fraction (measured by the biplane Simpson's method) were recorded in detail in the left recumbent position. TEE was examined by PhilipsiE33 color Doppler ultrasonography with an X7-2t transesophageal matrix real-time three-dimensional probe with a frequency of 2-7 MHz. Before fasting for 6-8 h, the ECG was recorded synchronously, the pulsed Doppler sampling volume was placed within 1 pulse near the opening of the left atrial appendage, and the blood flow spectrum of the left atrial appendage was obtained. The left atrial appendage blood flow spectrum was a regular two-way wave in sinus rhythm and an irregular zigzag waveform in atrial fibrillation rhythm. The peak value of the positive wave, that is, the maximum emptying velocity of the left atrial appendage, was recorded in 3 and 10 cardiac cycles, respectively, and the average value was taken as the velocity of the left atrial appendage (LAA-FV) ( Fig. 1-2).   was used to obtain CT imaging data. Iohexol (60-80 ml) was injected into the elbow vein at a flow rate of 5 ml/s, and then saline (50 ml) was injected at a speed of 5 ml/s. The contrast agent tracking technique triggered the enhanced scan. Image processing and measurement: All images were reconstructed by retrospective ECG gated reconstruction, slice thickness 0.5 mm, overlapping 0.3 mm, and image postprocessing was performed by an Advantage Workstation 3.2 (GE, USA) workstation. The CT threshold range of the adipose tissue was set to − 50 HU ~ − 200 HU. The left atrial epicardial adipose tissue (LA-EAT) was manually segmented, and the LA-EAT was calculated (Fig. 3). Three-dimensional images of the left atrial appendage and left atrium were obtained by three-dimensional reconstruction of the volume. According to the morphological characteristics of the left atrial appendage, the left atrial appendage was divided into three types: chicken wing type (chicken wing), obvious folding in the proximal or middle part of the main lobe of the left atrial appendage, and nonchicken wing type (nonchicken wing), excluding other forms of chicken wing type. The left atrial volume (LAV) and left atrial volume index (LAVI) were calculated, defined as the left atrial volume divided by the body surface area. All images were measured and evaluated by two independent clinicians using the same measurement methods, and any inconsistencies were resolved through consultation.
Detection of the low voltage zone (LVZ). All patients first underwent circumferential pulmonary vein isolation (PVI). Left atrial voltage mapping was performed directly in patients with paroxysmal atrial fibrillation after PVI. Patients with persistent atrial fibrillation underwent synchronous electrocardioversion if they were still in atrial fibrillation after isolation, and voltage mapping was performed after cardioversion. Voltage mapping was carried out with an ST ablation catheter (Johnson Company, USA), and contact force values of > 5 g for mapping electrograms confirmed adequate endocardial contact. We used ≥ 3 adjacent peak-to-peak bipolar voltage cutoffs of 0.2-0.5 mV to define the low-voltage region to more accurately delineate the LAVI 19 (Fig. 4).

Statistical analysis.
If the measurement data were normally distributed, they were expressed as the mean ± SD. If they do not conform to a normal distribution, they are expressed by the median (quartile spacing), that is, M (Q25 Q75), and the counting data are expressed by the number (percentage). The counting data were compared by Student's t test or Wilcoxon's singed rank-sum tests (nonnormal distribution), and the classified variables were compared by chi-square tests or Fisher's exact test. Pearson or Spearman correlation tests were used to evaluate the relationship between LAA-FV and various parameters. In addition, to evaluate the independent predictors of LAA-FV, multiple regression analysis was carried out. P < 0.05 was considered to be statistically significant. All statistical analyses were carried out using SPSS version 24.0 software (SPSS, Chicago, Illinois, USA).

Ethics declarations. Patient studies were approved by the Ethics Committee of Affiliated Hospital of
Xuzhou Medical University. All methods were implemented in accordance with relevant guidelines and regulations, and all enrolled patients signed informed consent forms.    www.nature.com/scientificreports/ shown in Table 2. Univariate analysis showed that the age of the LVZ group was older than that of the non-LVZ group. There were significant differences in terms of history of diabetes, left atrial volume index (LAVI), LA-EAT volume and left atrial appendage velocity (LAA-FV) between the two groups. In the LVZ group, there was more nonparoxysmal atrial fibrillation (P < 0.1), and the duration of atrial fibrillation was longer (P < 0.1). There was no significant difference in any other clinical or conventional echocardiographic parameters, including left ventricular ejection fraction, between the two groups (P > 0.05, Table 2).

Discussion
In recent years, increasing attention has been given to the relationship between epicardial adipose tissue and atrial fibrillation (AF). Venteclef et al. 20 reported that human adventitia adipose tissue (EAT) caused fibrosis of atrial cardiomyocytes in rats and promoted fibroblasts to differentiate into myofibroblasts. At present, the clinical detection methods for myocardial fibrosis are limited. Myocardial fibrosis is mainly detected through endocardial biopsy, late gadolinium enhancement cardiac magnetic resonance (LGE-CMR) and intracardiac voltage mapping. In this study, left atrial voltage mapping was performed during radiofrequency ablation, and patients with atrial fibrillation were divided into an LVZ group and a non-LVZ group. Compared with the non-LVZ group, the LAA-FV in the LVZ group was lower. This is consistent with the results of previous studies that show that there may be a correlation between atrial fibrosis and left atrial appendage velocity 13,14 . At present, TEE is the most widely used method to measure the function of the LAA, and it is considered the gold standard, but it is semi-invasive. We hope to find a non-invasive and reliable index to evaluate left atrial appendage function. In this study, it was found that left atrial LVZ, LA-EAT volume, left atrial appendage shape, left atrial volume index and type of atrial fibrillation (paroxysmal vs. persistence) were independent predictors of LAA-FV. Yuichi Hori et al. 21 found that patients with low LAA-FV (20 cm/s) were characterized by the presence of left anterior wall LVZ and high CHA 2 DS 2 VASc scores. SHIH-HSIENSUNG et al. 22 found that the larger the left atrium was, the larger the LVZ of the left atrium, and there was a significant correlation between the LVZ of the left atrium and the emptying velocity of the LAA. These results are consistent with our research. A possible reason is that atrial structural remodeling characterized by atrial fibrosis is the core of the maintenance mechanism of atrial fibrillation 23 . Left atrial electrical remodeling and fibrosis cause greater left atrial pressure, which is more likely to increase the afterload of the left atrial appendage and cause systolic dysfunction, therefore resulting in a decrease in LAA-FV. Another possible reason is that this structural remodeling also occurs in the left atrial appendage 24 . LAA-FV can reflect changes in left atrial function, including contraction, stunning, fibrosis, etc., so the LAA-FV may represent the severity of the left atrial remodeling 25 .
In a previous study, the types of atrial fibrillation were divided into paroxysmal atrial fibrillation, persistent atrial fibrillation and long-term persistent atrial fibrillation, and the results showed that LAA-FV decreased gradually 26 . The results of this study are consistent with the above studies, and the mechanism may be as follows: www.nature.com/scientificreports/ first, during TEE, the heart rhythm of patients with paroxysmal atrial fibrillation shows a sinus rhythm, while that of patients with persistent atrial fibrillation shows an atrial fibrillation rhythm. Thus, the emptying time of the left atrium and left atrial appendage is shortened, and rapid irregular electrical activity can significantly affect the active contractile force of the left atrial appendage muscle and decrease the velocity of the left atrial appendage. Previous studies have shown that LAAFV and LAA morphology are closely related. Lee JM et al. 27,28 found that the LAA orifice enlargement or a chicken wing type was closly related with the decreased flow velocity of LAA. The results of this study are consistent with the above studies. This may be because the chicken wing-type LAA has a higher muscle mass to contract the left atrial appendage 29 . Previous studies have shown that there is a significant negative correlation between the left atrial diameter and LAA-FV 30,31 . LAVI was used in this study because the use of the LA volume can more accurately identify structural atrial remodeling that involves the left atrial low-voltage matrix than the LA diameter. The results showed that LAVI was negatively correlated with LAA-FV and was an independent predictor of LAA-FV. The left atrial appendage is thought to act as a decompression chamber during left ventricular contraction and other increases in left atrial pressure 32 . LAA-FV may represent the full functionality of LA and indicate the severity of LA refactoring 33 . Therefore, dilatation of the left atrium and an increase in LAVI leads to remodeling of the left atrial appendage and a decrease in LAA-FV. This may also explain why some scholars believe that the risk of stroke increases with increasing left atrial size 34 .
A previous study 35 reported that the capacity of EAT may be related to the function of the left atrial appendage. However, that study did not investigate echocardiographic parameters. Yamaguchi et al. 36 evaluated the thickness of EAT by TTE. The results showed that the thickness of the EAT was negatively correlated with the filling and emptying velocity of the left atrial appendage. Because the thickness of the EAT varies greatly in different parts and the measurement repeatability is poor, this study improves the accuracy by measuring the EAT volume. It was found that the adipose tissue volume of the left atrium was an independent predictor of LAA-FV. The explanation may be as follows: First, EAT can cause atrial myocardial fibrosis through various pathways. Haemers et al. 37 proposed that EAT could lead to progressive fibrosis of the adjacent atrial myocardium through fatty infiltration. Venteclef et al. 20 found that EAT is the main source of activin A, and activin A may be the main factor mediating the pro-fibrotic effect of EAT. A recent study by Patel et al. 38 found that EAT can induce myocardial fibrosis by activating the renin-angiotensin-aldosterone system (RAAS), especially angiotensin II (Ang II). Previous studies [39][40][41] have confirmed that EAT can secrete inflammatory factors including interleukin-1 (IL-1), interleukin-6 (IL-6), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), nerve growth factor (NGF) and other inflammatory factors through endocrine and paracrine pathways. Under the action of these factors, myocardial inflammatory response is caused, which leads to myocardial fibrosis. As mentioned above, atrial fibrosis can lead to decreased LAAFV. Second, EAT is also closely related to oxidative stress. Salgado-Somoza et al. 42 obtained EAT from 55 patients undergoing cardiac surgery and detected oxidative stress indicators such as reactive oxygen species (ROS) in EAT tissue. The results suggest that EAT has higher levels of oxidative stress in patients with cardiovascular disease compared to other adipose tissues. Dudley et al. 43 established an atrial fibrillation model by rapid atrial pacing in pigs. The results showed that the level of oxidative stress in LAA tissues was significantly increased. Therefore, we speculate that the effect of EAT on LAA function may be related to oxidative stress. Oxidative stress can lead to cellular damage, impair myofibril energetics, and lead to myocardial contractile dysfunction, resulting in a decrease in LAAFV 44 .

Limitations
There are some limitations in this study. First, this study is a retrospective, single-center, small sample study, so it is necessary to conduct a prospective study on a larger cohort. Second, when measuring LA-EAT, semiautomatic methods are used to manually outline the epicardium, which may lead to differences in LA-EAT evaluation. In this study, two experienced imaging physicians separately performed the LA-AT measurement to reduce this measurement artifact. Third, the specific mechanisms of LA-EAT and LAA-FV are still unclear, such as EATinduced fibrosis, inflammation, and fat infiltration, which need to be studied further.

Conclusion
In NV-AF patients, the increase in LA-EAT is related to the decrease in LAA-FV. Currently, there are some limitations to CHA2DS2-VASc score to assess stroke risk in patients with NV-AF, which may be related to the fact that the score does not reflect the effect of cardiac structure and function on thrombosis. EAT may be able to compensate for the shortcomings of the CHA2DS2-VASc score.

Data availability
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.